Inhalational exposure to ozone and high concentration of oxygen can induce focal cell necrosis and sloughing of airway epithelial cells accompanied by recruitment of inflammatory cells, mucosal edema, increased mucus secretion and airway hyperresponsiveness. Tracheal epithelial (TE) cells are capable of generating various metabolites of arachidonic acid (AA) and may respond to oxidant gas exposure by altering the profile and production of these potent proinflammatory mediators. Necrosis and desquamation of airway epithelium can also cause increased release of neuropeptides such as Substance P from exposed afferent nerves and local generation of bradykinin from extravasated plasma. Receptors for Substance P and bradykinin are present on airway epithelial cells and these mediators may also activate AA metabolism by these cells at sites of airway injury. In preliminary studies we have observed alterations in the profile of AA metabolites generated by rabbit TE monolayers following in vitro exposure to ozone and hyperoxia as well as activation of AA cascade by Substance P and bradykinin. The objective of these studies is to examine in detail the effects of ozone and hyperoxic exposure on the production of AA metabolites by cultured rabbit and human TE cells and to determine the intracellular signalling mechanisms by which Substance P and bradykinin activate AA metabolism in airway epithelial cells. Monolayer cultures of rabbit and human TE cells maintained under conditions which allow for expression of endogenous eicosanoid metabolism will be exposed to clinically relevant concentrations of ozone and hyperoxia in a system which allows for in vitro exposure of cells at a gas-fluid interface analogous to in vivo conditions. Qualitative and quantitative changes in the spectrum of AA metabolites generated by TE cells in response to oxidant gas exposure and to Substance P and bradykinin will be assessed by high pressure signalling mechanisms linking peptide receptor coupling to activation of aA metabolism will be studied by measuring changes in free cytosolic calcium using the fluorescent indicator fura-2 and assessing G protein signal transduction, and the relative contributions of each mechanisms to activation of AA cascade determined by selective pharmacologic inhibition or stimulation of each signal pathway.